The overall goal of this procedure is to advance our capacity to protect individuals with antibodies or vaccines for preventing or treating histoplasmosis, and to examine the role of virulence factors in disease pathogenesis. This is accomplished by first isolating and characterizing monoclonal antibodies to target antigens. The second step of the procedure is to administer the selected monoclonal antibodies, intraperitoneal two hours prior to the intranasal infection.
In order for these molecules to have time to distribute throughout the body, the third step of the procedure is to anesthetize the mice and intranasally infect them with the desired inoculum. The final step of the procedure is to monitor the health of the animals. The animals are either euthanized at various intervals to determine fungal burdens and to assess inflammatory responses or are followed for survival.
Ultimately, results can be obtained that show a prolongation in survival and a modification of pathological responses in the affected organs of lethally infected mice Through peritoneal administration of the protective monoclonal antibodies prior to infection, Demonstrating the procedure will be ally emer, a postdoctoral student with extensive experience in fungal pathogenesis and animal infection models. The main advantage of this technique over existing methods like antifungal drugs, is that the monoclonal antibodies are molecules with ictal properties that work through engagement of the host potential factor. Response to disease visual demonstration of this method is critical.
As a monoclonal antibody administration and intranasal infection steps are difficult to learn because they require expertise in handling the animals. Prepare a biosafety level two cabinet for work with the fungus in the yeast form by cleaning the cabinet with bleach, followed by UV irradiation. Prepare a 15 milliliter conical tube with five milliliters of PBS.
Harvest a colony of H caps AUM from an aliquot of stock, frozen at minus 80 degrees Celsius, and add it to the PBS vortex. The cells vigorously and centrifuge for 1100 times, G for 10 minutes. At room temperature, carefully remove the supernatant without disturbing the pellet and add at least 10 milliliters of fresh PBS.
Repeat this procedure three times after the final wash. Resus resuspend the cells in five milliliters of PBS. All washes should be done at room temperature To disrupt aggregated cells, pass the yeast cell through a 26 gauge one half needle using a 10 milliliter syringe five times collecting the yeast cells in a 50 milliliter conical tube.
To isolate small, uniformly sized yeast, centrifuge the cells at 55 times G for one minute, and then remove the top one milliliter. Add the cell suspension to a sterile 250 milliliter glass erlenmeyer flask containing 100 milliliters of ham, F 12 medium, and then grow the cells at 37 degrees Celsius for 48 hours in an incubator with shaking at room carbon dioxide concentration. This culture will be used to prepare the inoculum and the efficacy of the monoclonal antibodies generated in the next step will be assessed.
Grow the selected hybridomas in DMEM after five to seven days. Incubation, harvest the antibodies by collecting all the media from the cell culture flask and then centrifuge the medium at 1100 times G for 10 minutes. As with the yeast cells, the antibodies may be washed at room temperature.
Collect the supernatant without disrupting the pellets. Next, purify the cell-free supernatant using a protein AG resin by F-P-L-C-A capture. Eli SA is used to calculate the monoclonal antibody concentration to determine how much of the purified antibody should be injected.
Intraperitoneal perform intraperitoneal administration of PBS Isotype control antibody or experimental antibody as described in the accompanying written protocol. Wait two hours after monoclonal antibody injection, before anesthetizing the mouse and proceeding with the intranasal infection during the waiting period. Prepare the histoplasma caps, lotum inoculum as described in the accompanying written protocol, and assemble the string apparatus for infection of the mouse by tightly tying a nylon or cotton string between two column stands and then separating the stands so that the string is tau.
The anesthesia ketamine at 100 milligrams per kilogram and xylazine at 10 milligrams per kilogram. Also can be prepped at this time when the antibodies have been given ample time to disperse. Anesthetize the mouse and confirm sedation by lack of response to toe.
Pinch by tweezers. Gently suspend the mouse by its front teeth to the string. Slowly administer approximately 50 microliters of inoculum into a air using a micropipet.
While closely monitoring the mouse's respiration rate. Allow the mouse to rest in this position for two to five minutes after intranasal infection to facilitate the deposition of the yeast into the animal's lungs after the mouse has been infected. Maintain the mouse in a monitored, warm environment carefully utilizing a heat lamp if necessary until it recovers from the anesthesia.
When the mouse awakens, return it to a clean cage. With ad libido, access to food and water assess infected and mock infected animals clinically. For tachypnea lethargy optimization and weight loss, the animals should be checked twice daily by laboratory members and daily by animal caretakers.
With murn histoplasmosis until close to the end of life, there are typically no apparent signs of the infection other than a mild increase in respiration rate. With advanced disease, the mice becomes significantly to kyp and have diminished activity. Typically, they rapidly become more abundant and expire.
Distressed animals should be euthanized appropriately with carbon dioxide. In a dedicated chamber. Confirmed death by lack of reflex response to toe, pinch to ceased animals should be enumerated daily.
Prepare 15 milliliter conical tubes with 10 milliliters of PBS for each organ to be collected. Euthanize animals infected with the sub lethal inoculum five times 10 to the sixth. The yeast cells as previously shown, and remove the lungs, spleens and livers immediately prepare 50 milliliter conical tubes with five milliliters of PBS.
Mount a Petri dish with 70 micron cell strainers for each organ to be homogenized. Macerate each organ separately with five milliliter plungers and transfer to 50 milliliter tubes. Next serially dilute the organ homogenate in three dilution at one to 100, one to 1001 to 5, 000 and plate 100 microliters on BHI.
Blood auger plates incubate the plates at 37 degrees Celsius in an incubator with normal room percentage of carbon dioxide for up to 14 days and enumerate the CFUs monoclonal antibody. Isotype is critical in protection against hcaps Lotum as mice treated with IgG one or IgG two a monoclonal antibodies to HS P 60 have significantly prolonged survival compared to control mice receiving an IgG two B monoclonal antibody to HSP 60 or either an isotype control monoclonal antibody or PS.Further in mice that received protective monoclonal antibodies, there was a significant reduction in the number of pulmonary and splenic CFUs at day seven and decreases of two and 2.5 log units of yeast numbers in the lungs at day 14. Following this procedure, other methods like determining ugal burden indicative of colony forming unity, the cytokines involved in the infection and evaluating the topology can be performed in order to answer additional questions like defining the mechanism of action of disease modification by the antibodies while attempting this procedure.
It's important to remember to work in biological safety conditions while handling the knock on and to work according to the animal care regulations for the internasal infections.
